EZH2 variants differentially regulate polycomb repressive complex 2 in histone methylation and cell differentiation

Abstract Background Polycomb repressive complex 2 (PRC2) is responsible for establishing and maintaining histone H3K27 methylation during cell differentiation and proliferation. H3K27 can be mono-, di-, or trimethylated, resulting in differential gene regulation. However, it remains unknown how PRC2 specifies the degree and biological effects of H3K27 methylation within a given cellular context. One way to determine PRC2 specificity may be through alternative splicing of Ezh2, PRC2’s catalytic subunit, during cell differentiation and tissue maturation. Results We fully characterized the alternative splicing of Ezh2 in somatic cells and male germ cells and found that Ezh’s exon 14 was differentially regulated during mitosis and meiosis. The Ezh2 isoform containing exon 14 (ex14-Ezh2) is upregulated during cell cycle progression, consistent with a role in maintaining H3K27 methylation during chromatin replication. In contrast, the isoform lacking exon 14 (ex14D-Ezh2) was almost exclusively present in spermatocytes when new H3K27me2 is established during meiotic differentiation. Moreover, Ezh2’s transcript is normally controlled by E2F transcription activators, but in spermatocytes, Ezh2’s transcription is controlled by the meiotic regulator MYBL1. Compared to ex14-EZH2, ex14D-EZH2 has a diminished efficiency for catalyzing H3K27me3 and promotes embryonic stem cell differentiation. Conclusions Ezh2’s expression is regulated at transcriptional and post-transcriptional levels in a cellular context-dependent manner. EZH2 variants determine functional specificity of PRC2 in histone methylation during cell proliferation and differentiation

Repression of the soma-specific transcriptome by Polycomb-repressive complex 2 promotes male germ cell development

Polycomb-repressive complex 2 (PRC2) catalyzes the methylation of histone H3 Lys27 (H3K27) and functions as a critical epigenetic regulator of both stem cell pluripotency and somatic differentiation, but its role in male germ cell development is unknown. Using conditional mutagenesis to remove the core PRC2 subunits EED and SUZ12 during male germ cell development, we identified a requirement for PRC2 in both mitotic and meiotic germ cells. We observed a paucity of mutant spermatogonial stem cells (SSCs), which appears independent of repression of the known cell cycle inhibitors Ink4a/Ink4b/Arf. Moreover, mutant spermatocytes exhibited ectopic expression of somatic lamins and an abnormal distribution of SUN1 proteins on the nuclear envelope. These defects were coincident with abnormal chromosome dynamics, affecting homologous chromosome pairing and synapsis. We observed acquisition of H3K27me3 on stage-specific genes during meiotic progression, indicating a requirement for PRC2 in regulating the meiotic transcriptional program. Together, these data demonstrate that transcriptional repression of soma-specific genes by PRC2 facilitates homeostasis and differentiation during mammalian spermatogenesis

Key mediators of somatic ATR signaling localize to unpaired chromosomes in spermatocytes

Meiotic silencing of unpaired chromatin (MSUC) occurs during the first meiotic prophase, as chromosomes that fail to pair are sequestered into a transcriptionally repressive nuclear domain. This phenomenon is exemplified by the heterologous sex chromosomes of male mammals, where the ATR DNA damage response kinase is crucial for this silencing event. However, the mechanisms underlying the initiation of MSUC remain unknown. Here, we show that essential components of ATR signaling in murine somatic cells are spatially confined to unpaired chromosomes in spermatocytes, including the ATR-dependent phosphorylation of the single-stranded DNA (ssDNA)-binding complex replication protein A (RPA) and the checkpoint kinase CHK1. These observations support a model in which ssDNA plays a central role in the recruitment of ATR during MSUC, and provide a link to meiotic progression through activation of CHK1

Differentiation-Driven Nucleolar Association of the Mouse Imprinted Kcnq1 Locus

The organization of the genome within the mammalian nucleus is nonrandom, with physiologic processes often concentrated in specific three-dimensional domains. This organization may be functionally related to gene regulation and, as such, may play a role in normal development and human disease processes. However, the mechanisms that participate in nuclear organization are poorly understood. Here, we present data characterizing localization of the imprinted Kcnq1 alleles. We show that nucleolar association of the paternal allele (1) is stimulated during the differentiation of trophoblast stem cells, (ii) is dependent upon the Kcnq1ot1 noncoding RNA, (3) does not require polycomb repressive complex 2, and (4) is not sufficient to preclude transcription of imprinted genes. Although nucleolar positioning has been proposed as a mechanism to related to gene silencing, we find that silencing and perinucleolar localization through the Kcnq1ot1 noncoding RNA are separable events

High resolution mapping and positional cloning of ENU-induced mutations in the Rw region of mouse chromosome 5

<p>Abstract</p> <p>Background</p> <p>Forward genetic screens in mice provide an unbiased means to identify genes and other functional genetic elements in the genome. Previously, a large scale ENU mutagenesis screen was conducted to query the functional content of a ~50 Mb region of the mouse genome on proximal Chr 5. The majority of phenotypic mutants recovered were embryonic lethals.</p> <p>Results</p> <p>We report the high resolution genetic mapping, complementation analyses, and positional cloning of mutations in the target region. The collection of identified alleles include several with known or presumed functions for which no mutant models have been reported (<it>Tbc1d14</it>, <it>Nol14</it>, <it>Tyms</it>, <it>Cad</it>, <it>Fbxl5</it>, <it>Haus3</it>), and mutations in genes we or others previously reported (<it>Tapt1</it>, <it>Rest</it>, <it>Ugdh</it>, <it>Paxip1</it>, <it>Hmx1, Otoe, Nsun7</it>). We also confirmed the causative nature of a homeotic mutation with a targeted allele, mapped a lethal mutation to a large gene desert, and localized a spermiogenesis mutation to a region in which no annotated genes have coding mutations. The mutation in <it>Tbc1d14 </it>provides the first implication of a critical developmental role for RAB-GAP-mediated protein transport in early embryogenesis.</p> <p>Conclusion</p> <p>This collection of alleles contributes to the goal of assigning biological functions to all known genes, as well as identifying novel functional elements that would be missed by reverse genetic approaches.</p

An Allelic Series Uncovers Novel Roles of the BRCT Domain-Containing Protein PTIP in Mouse Embryonic Vascular Development▿

Pax transactivation domain-interacting protein (PTIP, or PAXIP1) is required for mouse development and has been implicated in DNA damage responses and histone modification. However, the physiological roles of PTIP during embryogenesis remain unclear due to early embryonic lethality of null mutants. We describe two N-ethyl N-nitrosourea-induced hypomorphic missense alleles of Ptip, each of which alters one of the six encoded BRCT domains. Phenotypic characterization of these mutants revealed important functions of PTIP in vasculogenesis and chorioplacental development that appear unrelated to activities in DNA repair or global histone modification. The results of gene expression profiling and in vitro angiogenesis assays indicated that PTIP modulates a transcriptional program, centered around Vegfa, that drives the migration of endothelial cells to properly form the embryonic vasculature. These and other data suggest that PTIP has multiple functions, one of which is to promote the formation of transcriptional complexes that provide specificity of developmental gene expression

EZH2 variants differentially regulate polycomb repressive complex 2 in histone methylation and cell differentiation

Abstract Background Polycomb repressive complex 2 (PRC2) is responsible for establishing and maintaining histone H3K27 methylation during cell differentiation and proliferation. H3K27 can be mono-, di-, or trimethylated, resulting in differential gene regulation. However, it remains unknown how PRC2 specifies the degree and biological effects of H3K27 methylation within a given cellular context. One way to determine PRC2 specificity may be through alternative splicing of Ezh2, PRC2’s catalytic subunit, during cell differentiation and tissue maturation. Results We fully characterized the alternative splicing of Ezh2 in somatic cells and male germ cells and found that Ezh’s exon 14 was differentially regulated during mitosis and meiosis. The Ezh2 isoform containing exon 14 (ex14-Ezh2) is upregulated during cell cycle progression, consistent with a role in maintaining H3K27 methylation during chromatin replication. In contrast, the isoform lacking exon 14 (ex14D-Ezh2) was almost exclusively present in spermatocytes when new H3K27me2 is established during meiotic differentiation. Moreover, Ezh2’s transcript is normally controlled by E2F transcription activators, but in spermatocytes, Ezh2’s transcription is controlled by the meiotic regulator MYBL1. Compared to ex14-EZH2, ex14D-EZH2 has a diminished efficiency for catalyzing H3K27me3 and promotes embryonic stem cell differentiation. Conclusions Ezh2’s expression is regulated at transcriptional and post-transcriptional levels in a cellular context-dependent manner. EZH2 variants determine functional specificity of PRC2 in histone methylation during cell proliferation and differentiation